Doxycycline-induced inhibition of prolidase activity in human skin fibroblasts and its involvement in impaired collagen biosynthesis

Several lines of evidence suggest that doxycycline, a semi-synthetic deriva tive of tetracycline, may be a useful agent in the treatment of osteoarthri tis. It inhibits collagen synthesis and collagenase activity in hypertrophi c chondrocytes, slowing the process of collagen turnover. However, the mech anism of doxycycline-induced inhibition of these processes has not been est ablished. We considered prolidase, an enzyme involved in collagen metabolis m, as a possible target for the doxycycline-induced inhibition of collagen synthesis. Cultured human skin fibroblasts, specialized for collagen synthe sis, were used as model cells. Prolidase [E.C. 3.4.13.9] is a manganese-dep endent cytosolic exopeptidase that cleaves imidodipeptides containing C-ter minal proline, thus providing large amounts of proline for collagen resynth esis. Enzyme activity is regulated through the beta (1) integrin receptor. Therefore, we compared the effect of doxycycline on prolidase activity and expression, collagen biosynthesis, gelatinolytic activity and beta (1) inte grin expression in 24-h treated cultured human skin fibroblasts. We found t hat doxycycline induced coordinately inhibition of prolidase activity and c ollagen biosynthesis (IC50) at about 150 mug/ml) and gelatinolytic activity in cultured human skin fibroblasts. The inhibitory effect of doxycycline o n the processes was not due to the cytotoxicity of this drug, as shown in t he cell viability tetrazoline test. However, an inhibitory effect of the dr ug on DNA synthesis was observed (IC50 at about 100 mug/ml). The decrease i n prolidase activity in fibroblasts treated with doxycycline was not accomp anied by any differences in the amount of prolidase or beta (1) integrin re covered from these cells, as shown by Western immunoblot analysis. This sug gests that the doxycycline-induced down-regulation of prolidase is a post-t ranslational event. The data presented here raise the possibility that the doxycycline-induced decrease in collagen biosynthesis is mostly due to the inhibition of prolidase activity. (C) 2001 Elsevier Science B.V. All rights reserved.